Difference between revisions of "Part:BBa K3930014"
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<h2>Introduction</h2> | <h2>Introduction</h2> | ||
| − | <p>The promoter TetO7 is | + | <p>The promoter TetO7 is a doxycycline-inducible promoter. It must be used with the part coding for the activator protein rtTA advanced <a href="https://parts.igem.org/Part:BBa_K3930019" class="pr-0" target="_blank">(BBa K3930019)</a>. The sequence comes from the publication Garí et al. (1997).</p> |
<h2>Results</h2> | <h2>Results</h2> | ||
| − | <h3>Production of β-carotene | + | <h3>Production of β-carotene </h3> |
| − | <p> All the experiments that characterized this part are related to the final construct<a href="https://parts.igem.org/Part: | + | <p> All the experiments that characterized this part are related to the final construct pFRAMBOISE-notfused <a href="https://parts.igem.org/Part:BBa_K3930002" class="pr-0" target="_blank">(BBa K3930002)</a>, which was cloned into the <i>S. cerevisiae</i> LycoYeast strain. For more information on the experimental background, please refer to this part.</p> |
| − | <p>The promoter controls in the pFRAMBOISE-notfused construct the expression of the CrtY gene, which converts lycopene to β-carotene. The production of β-carotene is therefore a control of the functionality when the expression is induced with doxycycline. The carotenoids | + | <p>The promoter controls in the pFRAMBOISE-notfused construct the expression of the <i>CrtY</i> gene, which converts lycopene to β-carotene. The production of β-carotene is therefore a control of the functionality when the expression is induced with doxycycline. The carotenoids are contained in the cells, they were so extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after lysis was analyzed by RP-HPLC on a C18 column. In the LycoYeast-pFRAMBOISE-notfused strains (which express the CrtY), lycopene is converted into a new product with a higher retention time (Figure 1). Considering the β-ionone production results, we concluded this new peak most likely corresponds to β-carotene. Nonetheless, the peak is also observed in the condition with no induction. It seems like there is no negative regulation of the promoter TetO7.</p> |
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<b>Figure 1: </b> <b> Production of β-carotene upon doxycycline activation</b> | <b>Figure 1: </b> <b> Production of β-carotene upon doxycycline activation</b> | ||
| − | <p>β- | + | <p>β-carotene is produced <i>in vivo</i> by our strain. On the right are presented the mass spectra matching between the standard and the observed peak.</p> |
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| − | <p><b> | + | <p><b>We concluded this TetO7 promoter is not negatively regulating our gene expression and seems always active under those lab conditions.</b><p> |
<br> | <br> | ||
<h2>References</h2> | <h2>References</h2> | ||
Latest revision as of 08:55, 17 October 2021
Doxycycline inducible promoter
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 282
Illegal XhoI site found at 239 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
The promoter TetO7 is a doxycycline-inducible promoter. It must be used with the part coding for the activator protein rtTA advanced (BBa K3930019). The sequence comes from the publication Garí et al. (1997).
Results
Production of β-carotene
All the experiments that characterized this part are related to the final construct pFRAMBOISE-notfused (BBa K3930002), which was cloned into the S. cerevisiae LycoYeast strain. For more information on the experimental background, please refer to this part.
The promoter controls in the pFRAMBOISE-notfused construct the expression of the CrtY gene, which converts lycopene to β-carotene. The production of β-carotene is therefore a control of the functionality when the expression is induced with doxycycline. The carotenoids are contained in the cells, they were so extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after lysis was analyzed by RP-HPLC on a C18 column. In the LycoYeast-pFRAMBOISE-notfused strains (which express the CrtY), lycopene is converted into a new product with a higher retention time (Figure 1). Considering the β-ionone production results, we concluded this new peak most likely corresponds to β-carotene. Nonetheless, the peak is also observed in the condition with no induction. It seems like there is no negative regulation of the promoter TetO7.
We concluded this TetO7 promoter is not negatively regulating our gene expression and seems always active under those lab conditions.
References
- Garí E, Piedrafita L, Aldea M, Herrero E. 1997. A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast. 13(9):837–848. doi:10.1002/(SICI)1097-0061(199707)13:9<837::AID-YEA145>3.0.CO;2-T.